Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies

CRB2 is an essential protein of the renal filtration barrier. This study reports the development of an in vitro assay, suitable to investigate the pathogenic potential of known and novel, so far uncharacterized CRB2 variants.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** ---------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): 1. The authors follow up on their interesting studies on the pathogenic potential of CRB2 variants for an intact slit diaphragm and consequences of disruption of CRB2 function for the glomerular filter. They now provide additional evidence for deleteriousness of CRB2 missende variants by demonstrating significantly impaired intracellular trafficking of both variants described in patients and yet undescribed variants. These findings are of relevance for better understanding the function of CRB2 for the podocyte/slit diaphragm function and the general disease relevance of impaired trafficking of SD proteins.
2. The presented data and the choice for conducted experiments is clear and concise. We do understand the choice of easy to handle and transfect HEK293 cells. However, it would strengthen the data to repeat a subset of the tested variants (for instance one benign, one intermediate and one deleterious variant) in immortalized podocytes by transient transfection of the variant and labeling of the PM.
3. There are multiple minor issues to be addressed: -The authors should carefully reevaluate the use of the words 'variant' and 'mutation' -page 3: should say: publicly available databases - Fig 1 D: As authors have categorized into 3 groups, these should be visualized in the graph, potentially by displaying the partial group as yellow.
-page 5 bottom: should say: less severely affected -page 6 top: Fig1A -> Fig1B -page 7 first paragraph: revise truncated sentence that ends with 'includes several pathologically.' -include in discussion if impaired post-translational modifications and/or trafficking could be ameliorated by drugs and if respective studies are planned for future studies.
1st Authors' Response to Reviewers December 8, 2022 Reviewer #1 (Comments to the Authors (Required)): 1. The authors follow up on their interesting studies on the pathogenic potential of CRB2 variants for an intact slit diaphragm and consequences of disruption of CRB2 function for the glomerular filter. They now provide additional evidence for deleteriousness of CRB2 missense variants by demonstrating significantly impaired intracellular trafficking of both variants described in patients and yet undescribed variants. These findings are of relevance for better understanding the function of CRB2 for the podocyte/slit diaphragm function and the general disease relevance of impaired trafficking of SD proteins.
Comment 1: We are very happy that the reviewer find our data interesting and of relevance for the field of podocyte /slit diaphragm function and disease in the context of CRB2.
2. The presented data and the choice for conducted experiments is clear and concise. We do understand the choice of easy to handle and transfect HEK293 cells. However, it would strengthen the data to repeat a subset of the tested variants (for instance one benign, one intermediate and one deleterious variant) in immortalized podocytes by transient transfection of the variant and labeling of the PM.
Comment 2: We thank the reviewer for that comment and agree, showing the phenotypes in an additional cell culture model preferably immortalized podocytes will strengthen the presented data. However, there are some technical issues that should be considered: The mentioned HEK293 cell based assay system was established by a two-step process. In a first step we generated stable HEK293T cell lines expressing permanently the blue-fluorescent protein fused to the CAAX prenylation motif (BFP-CAAX). For that we used a retroviralbased system (RetroX) in combination with a pQCXIP expression vector that contains the BFP-CAX expression cassette. The transduced cells were selected by puromycin (because in addition to the gene of interest the pQCXIP vector backbone also mediates puromycin-resistance). In a second step we transiently transfected the HEK293T-BFP-CAAX cell lines with pINDUCER21_Puro plasmids. These plasmids encode for the different GFP-tagged CRB2 variants and induced its expression by the administration doxycycline. As HEK293 cells are easy to transfect with high transfection levels the system leads to robust and quantifiable results. In principle we can follow a similar strategy for immortalized podocytes, but immortalized podocytes are very difficult to handle and show very low transfection rates, especially for transient transfections. (Indeed, to avoid this problem and establish a fast possibility to screen CRB2 variants was the main reason, why we established a HEK293T-bassed cell assays system.) Nonetheless, we tried several approaches to transiently transfected immortalized podocytes by using different transfection agents, but all these approaches failed. Due to the very low transfection levels we obtained almost no cases in which two neighboring cells were positive for the expression of GFP-tagged CRB2 variants, which is essential for the evaluation of the CRB2 signal accumulating at the PM.
We also tried a sequential transduction strategy, by using first recombinant retrovirus (for BFP-CAAX) and next recombinant lentivirus particles (for GFP CRB2 expression, as described earlier by Möller-Kerutt et al., 2021). This approaches failed, as both recombinant viruses transfer a puromycin-resistance. In other words: clones selected by puromycin did not necessarily carry and express both genes of interest, BFP-CAAX and GFP-CRB2.
So we next tried a simultaneous transduction of podocytes with recombinant virusmixtures, encoding for BFP-CAAX (retrovirus) and GFP-CRB2 fusion proteins (lentivirus).
Here we obtained cell that expressed either BFP-CAAX, or GFP-labeled CRB2 or both (albeit in different ratios).
The images illustrate this problem in more detail. So finally, to address the reviewer's points and to confirm obtained results of the HEK293 system (in Fig. 1) we decide to follow the strategy of the foregoing study (Möller-Kerutt et al., 2021, JASN;Fig. 6 and suppl. Fig. S6). Here, we did the evaluation without a blue labelled plasma membrane and evaluated CRB2-GFP positive overlapping regions between two cells and used and ER labeling as reference in human immortalized podocytes.
By using this setting, we analyzed the localization of the wildtype-like M145T CRB2 variant, the disease-associated C620S CRB2 variant and a variant with an intermediate cellular phenotype (R1072 CRB2 variant). Quantification according to the protocol of Möller-Kerutt et al revealed that these CRB2 variants showed a same trend as observed in HEK293T cells. We included these data as novel suppl. Fig. to the manuscript. 3. There are multiple minor issues to be addressed: -The authors should carefully reevaluate the use of the words 'variant' and 'mutation' Comment 3: We thank the reviewer for this comment. Indeed, from geneticists it is sometimes recommended to avoided the word mutants, 1 st , as it not always clear at what point a variants becomes a (disease associated or -causing) mutant, and 2 nd as even in case that variants are disease-associated it is not clear in how far their penetrance is also influence by additional factors. Consequently, we replaced the word "mutants" by the "variants", except for the artificial deletion mutants lacking the 10 th EGF repeat (∆EGF 10). (Thus, variants that cause diseases are now called disease-associated variants, in cases where this it is not clear we call them putative disease-associated variants.) -page 3: should say: publicly available databases Comment 4: We changed this term as recommended.
- Fig 1 D: As authors have categorized into 3 groups, these should be visualized in the graph, potentially by displaying the partial group as yellow.
Comment 5: In the Quantification in Fig. 1D we evaluated the CRB2-GFP between two GFP-positive cells, whether it is at the PM or not, independent of the degree of PM localization (whether it is full or partial). Differences in the degree of PM localization were assayed by co-localization analysis summarized in Fig. 1E.
To address reviewers point we underlined the CRB2 variants in the figure that represent the intermediate group.
-page 5 bottom: should say: less severely affected Comment 6: We changed this term as recommended. Comment 7: Thanks for this hint! We changed this as recommended.
-page 7 first paragraph: revise truncated sentence that ends with 'includes several pathologically.' Comment 8: We now changed the truncated sentence into: "We also designed an artificial deletion mutant lacking the complete 10 th EGF-repeat (aa 605-640) that includes several variants with known are anticipated pathological potential (Fig. 1B)." -include in discussion if impaired post-translational modifications and/or trafficking could be ameliorated by drugs and if respective studies are planned for future studies.
Comment 9: We thank the reviewer for this comment: Indeed, in vitro system could be useful for high-throughput experiments, planned to the therapeutic potential of agents and drugs (including for example chemical chaperones). Therefore we added the passage: "How disturbances and delays of the SD processing and posttranslational modifications contribute to human inherited forms of SRNS-podocytopathies requires further analyses. The here described assay system is perhaps a suitable technical tool for testing of the pathological potential of novel uncharacterized CRB2 protein variants. In addition, it might be also serve as a cell-based platform for evaluating the therapeutic potential of agents (including for example chemical chaperones) influencing CRB2 folding and export to the cell surface or the SD." Prof. Thomas Weide University Hospital Münster Molecular Nephrology Albert-Schweitzer-Campus 1, A14 Muenster D-48149 Germany Dear Dr. Weide, Thank you for submitting your revised manuscript entitled "Loss-of-surface-transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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